PROJECT SUMMARY/ABSTRACT Each year, up to 50,000 patients in the United States receive unnecessary thyroidectomies occurring due to the difficulty in pre-operative distinguishing benign thyroid nodules from thyroid cancers. Thyroidectomy may result in the lifelong need for thyroid hormone replacement and calcium supplementation, and possibly a permanently hoarse voice. The objective of this study is to develop and validate a new epigenetic test to improve thyroid cancer diagnostics. Using published current molecular diagnostic tests, over half of thyroid nodules that are predicted to have significant cancer risk are, when surgically excised, found to be benign (~60% failure rate). Current molecular methods have focused on developing molecular diagnostics based on the difference between thyroid cancer and normal thyroid tissue. This approach is problematic because in contrast to normal thyroid tissues, benign thyroid nodules can contain cancer associated molecular alterations like mutations and gene fusions. We have instead focused equal attention to the difference between benign thyroid nodules and normal thyroid tissue and developed a novel DNA methylation-based diagnostic approach. We performed pilot studies in 55 thyroid nodules using a cross-validation approach. These initial studies show that our epigenetic test can differentiate benign thyroid nodules versus thyroid cancer with an estimated sensitivity of 94% and specificity of 100%. Here we propose to further assess and validate the ability of our DNA methylation markers to differentiate benign thyroid nodules versus thyroid cancer. Guided by our preliminary data we hypothesize that a DNA methylation based approach (i) can be successfully used for molecular thyroid cancer diagnostics and (ii) have a superior performance in comparison to current thyroid cancer diagnostic testing (~60% failure rate). The specific aims are: Specific Aim 1: To determine the diagnostic accuracy of the DNA methylation based approach for thyroid cancer diagnostics in retrospectively obtained 300 benign and 300 malignant thyroid nodules. Specific Aim 2: To compare the diagnostic accuracy between DNA methylation based approach and two thyroid cancer diagnostic approaches currently implemented in clinical practice by using retrospective cohort containing 600 thyroid nodules. This study is significant because it is expected that this diagnostic approach will supplant current diagnostics of thyroid nodules and decrease needless thyroid surgeries and the cost of medical care. This study is innovative because it emphasizes the difference between benign nodules and normal thyroid tissue and pioneers the use of DNA methylation for thyroid nodule diagnostics.